Ricardo Dorr

A new data-acquisition system for the measurement of the net water flux across epithelia

We describe a computer-based data-acquisition system designed for the measurement of net water flux across epithelia. It is based on the detection of a liquid meniscus position in a capillary tube using an electro-optical device. Then the data are digitized, transferred to the computer main memory and saved in a file. The advantages of the system are: (a) to measure net water transfer with an accuracy of 50 nl; (b) to switch from absorptive to secretory conditions in real time; (c) data are directly digitized to be analytically, graphically and statistically treated; (d) the specially developed software allows change of time intervals for data acquirement and the scale in graphic events; (e) the software also allows recall to the monitor screen of graphics from previous tests or to print them on paper; (f) a simple mechanical structure, saving maintenance and time; and (g) high reliability.

Uroguanylin Regulates Net Fluid Secretion via the NHE2 Isoform of the Na+/H+ Exchanger in an Intestinal Cellular Model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na+/H+ exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na+/H+ exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (Jv) was studied by optic techniques and intracellular pH (pHi) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na+ absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pHi. In the assayed conditions, cell exposure to apical UGN increases net secretory Jv, without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net Jv was produced mainly by a reduction in normal Na+ absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pHi is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pHi regulation.

Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins.

Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

Simple multichannel system for the measurement of the net water flux across biological tissues

This paper describes the development of a simple system for measurement of net water movement through biological membrane barriers. The system is based on the detection of a water meniscus inside a polyethylene tube, which reflects the water movement inside one hemichamber of a modified Ussing chamber containing a membrane barrier. The detection device consists of a commercial computer-controlled flat bed scanner and specifically developed software. This system allows one to perform a relatively high number of individual experiments per physical unit. It is a flexible and affordable device, which allows comparatively more information per unit to be obtained than previously described methods.

A counterpoint between computer simulations and biological experiments to train new members of a laboratory of physiological sciences

When new members join a working group dedicated to scientific research, several changes occur in the groups dynamics. From a teaching point of view, a subsequent challenge is to develop innovative strategies to train new staff members in creative thinking, which is the most complex and abstract skill in the cognitive domain according to Blooms revised taxonomy. In this sense, current technological and digital advances offer new possibilities in the field of education. Computer simulation and biological experiments can be used together as a combined tool for teaching and learning sometimes complex physiological and biophysical concepts. Moreover, creativity can be thought of as a social process that relies on interactions among staff members. In this regard, the acquisition of cognitive abilities coexists with the attainment of other skills from psychomotor and affective domains. Such dynamism in teaching and learning stimulates teamwork and encourages the integration of members of the working group. A practical example, based on the teaching of biophysical subjects such as osmosis, solute transport, and membrane permeability, which are crucial in understanding the physiological concept of homeostasis, is presented.

Fura-2 transport in toad urinary bladder epithelium: effects of antidiuretic hormone, colchicine and osmotic gradients

Fluorescence is transferred across the toad urinary bladder when fura-2/AM is added to the mucosal or serosal sides of the epithelium. It was now observed that: (1) Oxytocin (20 nM, serosal) increased fluorescence transfer from the mucosal to the serosal but not from the serosal to the mucosal baths. The ratio between the fluorescence intensities recorded with excitation wavelengths of 340 and 380 nm indicates that the calcium sensitive probe (free fura-2) was transferred to the serosal but not to the mucosal compartment by an oxytocin sensitive transport. (2) Preincubation with probenecid did not change fluorescence transfer in basal conditions but significantly reduced the oxytocin induced increase in free fura-2 transport. (3) Fluorescence accumulation inside the tissue was strongly reduced by oxytocin, but only when fura-2/AM was added to the mucosal side. (4) An osmotic gradient, in the presence of oxytocin, further increased the transfer of fluorescence at 380 nm but not at 340 nm. This indicated that the transfer of a calcium-insensitive fraction was being stimulated. (5) Preincubation with colchicine strongly inhibited fluorescence transfer across the tissue, at both 340 and 380 nm (the 340/380 ratio did not change). (6) Tissue accumulation was increased by colchicine. (7) Vanadate did not inhibit fura-2 transfer in the toad urinary bladder. We conclude that intracellularly-generated free fura-2 is only transported across the basolateral border, and that this transfer is stimulated by ADH. The calcium-insensitive fraction is transferred by a temperature-dependent process, sensitive to an osmotic gradient and colchicine.

Fura-2 handling in a polarized epithelial barrier: The toad urinary bladder

Toad bladders sacs were placed inside quartz cuvettes. When fura-2 AM was added to the mucosal compartment, low temperature (4 degrees C) almost completely blocked the transepithelial transfer of fluorescence observed at 20 degrees C (20 degrees C = 371 +/- 56, 4 degrees C = 29 +/- 29 fluorescence intensity in arbitrary units (FIAU), excitation at 340 nm, emission at 510 nm). Simultaneously, fluorescence accumulation inside the tissue was significantly higher (20 degrees C = 25 +/- 5, 4 degrees C = 91 +/- 24% increase on basal levels (%IBL)). When fura-2 AM was added to the serosal side, low temperature also reduced the serosal to mucosal transfer (20 degrees C = 149 +/- 36, 4 degrees C = 61 +/- 35 FIAU). Nevertheless, in this situation tissue accumulation, that was significantly higher that the one observed when fura-2 AM was added to the mucosal side, was reduced at low temperature (20 degrees C = 300 +/- 30, 4 degrees C = 48 +/- 7 %IBL). Spectral analysis of the mucosal and serosal compartments indicated that free fura-2 was transferred from the intracellular to the serosal compartment, but not to the mucosal one. These results indicate that fura-2 appears as a useful tool to evaluate the cellular distribution and traffic of polycyclic charged and non-charged molecules.